Process for preparing a sterile, dry crosslinking agent

ABSTRACT

The present invention discloses a novel method for preparing crosslinked biomaterial compositions for use in the augmentation of soft or hard tissue. In general, the method comprises mixing a biocompatible polymer, which is preferably collagen, with a sterile, dry crosslinking agent, which is preferably a synthetic hydrophilic polymer such as a functionally activated polyethylene glycol. Also provided are preferred processes for preparing sterile, dry crosslinking agents contained within syringes for use in the method of the invention. Methods for sterilization of the crosslinking agent include, but are not limited to, sterile filtration, aseptic processing, and e-beam or gamma irradiation. Methods for providing augmentation of soft or hard tissue using crosslinked biomaterial compositions prepared according to the method of the invention are also disclosed.

CROSS-REFERENCES

This application is a continuation-in-part of copending U.S. applicationSer. No. 08/287,549, filed Aug. 8, 1994, which is a continuation-in-partof U.S. application Ser. No. 08/236,769, filed May 2, 1994 and now U.S.Pat. No. 5,475,052 which is a continuation-in-part of U.S. applicationSer. No. 08/198,128, filed Feb. 17, 1994 and now U.S. Pat. No. 5,413,791which is a divisional of U.S. application Ser. No. 07/922,541 filed Jul.30, 1992 and now U.S. Pat. No. 5,328,955, issued Jul. 12, 1994, which isa continuation-in-part of U.S. application Ser. No. 07/433,441, filedNov. 19, 1989 U.S. Pat. No. 5,162,430, issued Nov. 10, 1992, which is acontinuation-in-part of U.S. application Ser. No. 07/274,071, filed Nov.21, 1988, subsequently abandoned, which applications and issued patentsare incorporated herein by reference in full, and to which currentlypending applications we claim priority under 35 U.S.C. §120.

FIELD OF THE INVENTION

In general, this invention relates to a method of preparing crosslinkedbiomaterial compositions, which preferably comprise collagen or otherbiocompatible polymer crosslinked using a synthetic hydrophilic polymer,for use in tissue augmentation and in the production of formed implantsfor various medical uses. The invention also provides an article ofmanufacture comprising a syringe containing a sterile, dry, crosslinkingagent and processes for preparing sterile, dry crosslinking agentscontained within syringes for use in the methods of the presentinvention.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 5,162,430, issued Nov. 10, 1992 to Rhee et al., andcommonly owned by the assignee of the present application, disclosescollagen-synthetic polymer conjugates and methods of covalently bindingcollagen to synthetic hydrophilic polymers. Commonly owned U.S. Pat. No.5,292,802, issued Mar. 8, 1994, discloses methods for making tubescomprising collagen-synthetic polymer conjugates. Commonly owned U.S.Pat. No. 5,306,500, issued Apr. 26, 1994, discloses methods ofaugmenting tissue with collagen-synthetic polymer conjugates.

Commonly owned U.S. Pat. No. 5,328,955, issued Jul. 12, 1994, disclosesvarious activated forms of polyethylene glycol and various linkageswhich can be used to produce collagen-synthetic polymer conjugateshaving a range of physical and chemical properties. Commonly owned,copending U.S. application Ser. No. 07/984,933, filed Dec. 2, 1992,discloses methods for coating implants with collagen-synthetic polymerconjugates.

Commonly owned, copending U.S. application Ser. No. 08/146,843, filedNov. 3, 1993, discloses conjugates comprising various species ofglycosaminoglycan covalently bound to synthetic hydrophilic polymers,which are optionally bound to collagen as well. Commonly owned,copending U.S. application serial No. 08/147,227, filed Nov. 3, 1993,discloses collagen-polymer conjugates comprising chemically modifiedcollagens such as, for example, succinylated collagen or methylatedcollagen, covalently bound to synthetic hydrophilic polymers to produceoptically clear materials for use in ophthalmic or other medicalapplications.

Commonly owned U.S. application Ser. No. 08/236,769, filed May 2, 1994,discloses collagen-synthetic polymer matrices prepared using a multiplestep reaction.

All publications cited above and herein are incorporated herein byreference to describe and disclose the subject matter for which it iscited.

In our earlier issued patents and applications described above, wedisclosed biomaterial compositions comprising collagen or otherbiocompatible polymers crosslinked using synthetic hydrophilic polymers.These crosslinked compositions were generally prepared by mixing aqueoussuspensions of collagen or biocompatible polymers with aqueous solutionsof synthetic hydrophilic polymers. The resulting crosslinked biomaterialcompositions could be used in a variety of medical applications, such assoft tissue augmentation and the preparation of biocompatibleimplantable devices.

Unfortunately, there was a major drawback to the method of preparingcrosslinked biomaterial compositions described above: synthetichydrophilic polymers, such as functionally activated polyethyleneglycols, are highly reactive with water, as well as with collagen andother polymers having corresponding reactive groups such as, for example(and not by way of limitation), available amino groups. The longer thesynthetic hydrophilic polymer is exposed to water (or water-basedcarriers), the more of its activity is lost due to hydrolysis, resultingin partial to complete loss of crosslinking ability. Therefore, in orderto avoid significant loss of crosslinking activity due to hydrolysis,the synthetic hydrophilic polymer must be thoroughly mixed with anaqueous carrier to prepare a homogeneous, aqueous crosslinker solutionimmediately prior to being mixed with an aqueous suspension of collagen(or other suitable biocompatible polymer) to prepare a crosslinkedbiomaterial composition. Unfortunately, a certain amount of activitycould still be expected to be lost, despite the speed of the operatorpreparing the composition.

While the above method for preparing crosslinked biomaterialscompositions had its drawbacks with respect to preparing formedimplants, it represented an even greater hurdle in the development of aviable commercial product for use in tissue augmentation. For example,the synthetic hydrophilic polymer could not be stored in an aqueousstate because it would hydrolyze, nor could it be stored mixed with thecollagen because the two components would react and form anon-extrudable gel within the syringe. Therefore, the synthetichydrophilic polymer needed to be provided to a physician in dry form,then dissolved in an aqueous carrier immediately prior to mixing withthe collagen suspension. The contemplated method required a number ofpreparatory steps that needed to be performed in rapid succession by thephysician in order to provide successful tissue augmentation. In otherwords, the suggested process was cumbersome and certainly not "userfriendly".

In the contemplated method, the physician would be provided with asyringe containing an appropriate amount of an aqueous carrier solution,such as phosphate-buffered saline (PBS), a vial containing anappropriate amount of a dry crosslinking agent, such as a synthetichydrophilic polymer, and a relatively large-gauge needle, such as a20-gauge needle. Prior to mixing the crosslinking agent with thecollagen (which would be provided in its own syringe), the physicianwould need to perform the following steps: 1) unwrap the packagecontaining the needle; 2) remove the cap from the syringe containing theaqueous carrier; 3) attach the needle to the syringe; 4) dispense theaqueous carrier by means of the needle into the vial containing the drycrosslinking agent; 5) vortex or otherwise adequately mix thecrosslinking agent with the aqueous carrier within the vial to producean aqueous crosslinker solution (which has already started to hydrolyzein the presence of water); 6) withdraw the crosslinker solution into thesyringe; and 7) remove the needle from the syringe in preparation formixing the crosslinker solution with the collagen (or otherbiomaterial). All of these preparatory steps would need to be performedwithin minutes of mixing the crosslinker solution with the collagen andinjecting the patient in order to minimize loss of crosslinker activity.

SUMMARY OF THE INVENTION

In situ crosslinking of biocompatible polymers and crosslinking agentsrequires that the crosslinking agent and biocompatible polymer beuniformly mixed for formation of a strong, cohesive implant followinginjection. At the time the method described above was developed, it wasnot contemplated that it would be possible to obtain adequate mixing ofcrosslinking agents in dry form with a biocompatible polymer to achievean evenly crosslinked implant. However, we have since discovered methodsof mixing dry crosslinking agents with aqueous suspensions of collagenwhich result in the production of strong, evenly crosslinked implants.In fact, our experiments indicate that uniform mixing is achievable whenaqueous suspensions of collagen are mixed with either dry crosslinkingagents or with aqueous solutions of crosslinking agents. Uniform mixingresults in crosslinked gels having good mechanical strength and lowspatial variability in mechanical strength. Non-uniform mixing can leadto local regions of low crosslinker concentration within the gel,resulting in lower average gel strengths and greater variability inmechanical strength spatially across the gel.

Our experiments indicate that, surprisingly, crosslinked gels producedusing dry crosslinking agents may have greater average strength, andless spatial variability in strength, than gels produced using aqueoussolutions of crosslinking agents. We have attributed this unexpectedresult to the possibility that, because dry crosslinking agents must besolubilized prior to reacting with collagen, they may be expected toreact more slowly with collagen than would aqueous crosslinking agents.Dry crosslinking agents must first be solubilized prior to reacting withcollagen, allowing additional time before the initiation of gel networkformation, during which time the crosslinking agent can be morehomogeneously mixed with the collagen. The faster reaction times ofaqueous crosslinking agents may lead to weaker gels because thecrosslinked gel may fracture if mixing is still in progress while gelnetwork formation is occurring.

An additional benefit exists for dry crosslinking agents which arehydrolytically unstable. When dry crosslinking agents are mixed withaqueous suspensions of collagen (or other biocompatible polymer), thereactive sites of the crosslinking agent are in contact with water for ashorter period of time before they can react with the collagen, whichmay result in greater crosslinking efficiency (crosslinking density)because the rate of aminolysis for crosslinking agents such as synthetichydrophilic polymers is always faster than their rate of hydrolysis(e.g., for a synthetic hydrophilic polymer such as difunctionallyactivated SG-PEG, the rate of aminolysis is at least ten times greaterthan the rate of hydrolysis).

The optimization of techniques for mixing biocompatible polymers and drycrosslinking agents led to the development of a method for providingtissue augmentation having a reduced number of steps compared to thepreviously contemplated method. The present invention provides a methodfor preparing crosslinked biomaterial compositions comprising: providinga means for delivering a biocompatible polymer and a means fordelivering a sterile, dry crosslinking agent; mixing the biocompatiblepolymer with the dry crosslinking agent to initiate crosslinking betweenthe biocompatible polymer and the crosslinking agent; and delivering thebiocompatible polymer and the crosslinking agent to a mold having thedesired size and shape. The present invention also provides a method foreffecting tissue augmentation that consists of a minimum number of stepsand is therefore very "user friendly" to the physician. Said methodcomprises the steps of: providing a means for delivering a biocompatiblepolymer and a means for delivering a sterile, dry crosslinking agent;mixing the biocompatible polymer with the dry crosslinking agent toinitiate crosslinking between the biocompatible polymer and thecrosslinking agent; and delivering the biocompatible polymer and thecrosslinking agent to the tissue site in need of augmentation.

Because the crosslinked biomaterial compositions prepared using themethod of the invention are destined to be injected or otherwiseimplanted into a human body, it is necessary that the dry crosslinkingagents be provided in a sterile form, and that they retain theirsterility over long-term storage. We therefore developed severalprocesses for preparing dry, sterile crosslinking agents containedwithin syringes.

One such process comprises dissolving a dry crosslinking agent in afluid carrier to produce a crosslinker solution, sterile-filtering thecrosslinker solution, dispensing the sterile crosslinker solution into asyringe, followed by lyophilizing the sterile crosslinker solutionwithin the syringe under aseptic conditions. A similar process comprisesdissolving a dry crosslinking agent in a fluid carrier to produce acrosslinker solution, dispensing the crosslinker solution into asyringe, lyophilizing the crosslinker solution within the syringe, thensterilizing the resulting dry crosslinking agent within the syringeusing irradiation.

Yet another process involves dissolving a dry crosslinking agent in anonaqueous carrier to produce a nonaqueous crosslinker solution,sterile-filtering the crosslinker solution, dispensing the sterilecrosslinker solution into a syringe, and then drying the sterilecrosslinking agent within the syringe by evaporation under asepticconditions. A similar process comprises dissolving a dry crosslinkingagent in a nonaqueous carrier to produce a nonaqueous crosslinkersolution, dispensing the crosslinker solution into a syringe, drying thecrosslinker solution within the syringe by evaporation, then sterilizingthe dry crosslinking agent within the syringe by irradiation.

An alternative process comprises dispensing a dry crosslinking agentinto a syringe, then sterilizing the dry crosslinking agent within thesyringe using irradiation. A particularly preferred embodiment of theaforementioned process comprises molding a dry crosslinking agent toform a pellet and sterilizing the pellet using e-beam irradiation

Sterile, dry crosslinking agents prepared as described above can bestored for long periods of time within a syringe (or other deliverymeans) while maintaining their activity (i.e., reactivity withbiocompatible polymers) as well as their sterility.

We now disclose a detailed description of preferred embodiments of thepresent invention, including improved methods for preparing crosslinkedbiomaterial compositions and for effecting tissue augmentation usingcrosslinked biomaterial compositions, as well as methods for providingsterile, dry crosslinking agents contained within syringes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of mechanical compression testing forcrosslinked collagen-gels having a 35 mg/ml collagen concentration and a5 mg/ml concentration of difunctionally activated SG-PEG (DSG-PEG)powder, which were prepared by mixing the collagen and dry DSG-PEG backand forth between two syringes, each having a barrel volume of 3 cc, fora total of 40 passes between the two syringes (3:3P40). The gels wereallowed to cure for 6 hours at 37° C., then sectioned into 4-mm thickdisks for mechanical testing. Samples were compressed to failure in theInstron Universal Tester, Model 4202, at a constant rate of 2millimeters per minute. Force (in Newtons) required to cause failure ofthe gel is graphed against position of the sample from the syringe tip.

DEFINITIONS

It must be noted that, as used in this specification and the appendedclaims, the singular forms "a", "an", and "the" include plural referentsunless the context clearly dictates otherwise. For example, reference to"a conjugate" includes one or more conjugate molecules, reference to "anarticle" includes one or more different types of articles known to thoseskilled in the art and reference to "the collagen" includes mixtures ofdifferent types of collagens and so forth.

Specific terminology of particular importance to the description of thepresent invention is defined below:

The term "aqueous carrier" refers to a water-based fluid carrier, suchas water-for-injection (WFI) or a solution of phosphate-buffered saline(PBS).

The term "atelopeptide collagen" refers to collagens which have beenchemically treated or otherwise processed to remove the telopeptideregions, which are known to be responsible for causing an immuneresponse in humans to collagens from other animal, such as bovine,sources.

The terms "chemically conjugated" and "conjugated" as used herein meanattached through a covalent chemical bond. In the practice of theinvention, a hydrophilic synthetic polymer and a biocompatible polymermolecule may be covalently conjugated directly to each other by means ofa functional group on the synthetic hydrophilic polymer, or thebiocompatible polymer and the synthetic polymer may be covalentlyconjugated using a linking radical, so that the hydrophilic syntheticpolymer and the biocompatible polymer are each bound to the radical, butnot directly to each other.

The term "collagen" as used herein refers to all types and forms ofcollagen, including those which have been recombinantly produced,extracted from naturally occurring sources (such as bovine corium orhuman placenta), processed, or otherwise modified.

The term "collagen-in-solution" or "CIS" refers to collagen in an acidicsolution having a pH of approximately 3 or less, such that the collagenis in the nonfibrillar form.

The term "collagen suspension" refers to a suspension of collagen fibersin an aqueous carrier, such as water or phosphate-buffered saline (PBS).

The term "collagen-synthetic polymer" refers to collagen chemicallyconjugated to a synthetic hydrophilic polymer, within the meaning ofthis invention. For example, "PEG-collagen" denotes a composition of theinvention wherein molecules of collagen are covalently conjugated tomolecules of polyethylene glycol (PEG).

"Crosslinked collagen" refers to a collagen composition in whichcollagen molecules are linked by covalent bonds with multifunctionallyactivated synthetic hydrophilic polymers, such as difunctionallyactivated polyethylene glycol.

The term "difunctionally activated" refers to synthetic hydrophilicpolymer molecules which have been chemically derivatized so as to havetwo functional groups capable of reacting with primary amino groups onbiocompatible polymer molecules, such as collagen or deacetylatedglycosaminoglycans. The two functional groups on a difunctionallyactivated synthetic hydrophilic polymer are generally located atopposite ends of the polymer chain. Each functionally activated group ona difunctionally activated synthetic hydrophilic polymer molecule iscapable of covalently binding with a biocompatible polymer molecule,thereby effecting crosslinking between the biocompatible polymermolecules.

The term "dry" means that substantially all unbound water has beenremoved from a material.

The term "fibrillar collagen" refers to collagens in which the triplehelical molecules aggregate to form thick fibers due to intermolecularcharge and hydrophobic interactions.

The term "fluid carrier" refers to a flowable carrier which may beeither an aqueous or a nonaqueous carrier, as specified.

The term "functionally activated" refers to synthetic hydrophilicpolymers which have been chemically derivatized so as to have one ormore functional group capable of reacting with primary amino groups onbiocompatible polymer molecules.

The term "in situ" as used herein means at the site of administration.

The term "in situ crosslinking" as used herein refers to crosslinking ofa biocompatible polymer implant following implantation to a tissue siteon a human or animal subject, wherein at least one functional group onthe synthetic polymer is covalently conjugated to a biocompatiblepolymer molecule in the implant, and at least one functional group onthe synthetic polymer is free to covalently bind with otherbiocompatible polymer molecules within the implant, or with collagenmolecules within the patient's own tissue. The term "lyophilized" refersto a dry state which has been achieved by freezing a wet substance andevaporating the resulting ice. Lyophilization is intended to preservewet substances. The product is frozen, then exposed to an atmosphere oflow relative humidity in which the ice contained within the productsublimes, i.e., transforms directly from a solid to a vapor withoutmelting. The necessary low relative humidity is generally achieved byconducting the process under a vacuum.

The term "molecular weight " as used herein refers to the weight averagemolecular weight of a number of molecules in any given sample, ascommonly used in the art. Thus, a sample of PEG 2000 might contain astatistical mixture of polymer molecules ranging in weight from, forexample, 1500 to 2500, with one molecule differing slightly from thenext over a range. Specification of a range of molecular weightindicates that the average molecular weight may be any value between thelimits specified, and may include molecules outside those limits. Thus,a molecular weight range of about 800 to about 20,000 indicates anaverage molecular weight of at least about 800, ranging up to about20,000.

The term "multifunctionally activated" refers to synthetic hydrophilicpolymers which have been chemically derivatized so as to have two ormore functional groups which are located at various sites along thepolymer chain and are capable of reacting with primary amino groups onbiocompatible polymer molecules. Each functional group on amultifunctionally activated synthetic hydrophilic polymer molecule iscapable of covalently binding with a biocompatible polymer molecule,thereby effecting crosslinking between the biocompatible polymermolecules. Types of multifunctionally activated hydrophilic syntheticpolymers include difunctionally activated, tetrafunctionally activated,and star-branched polymers.

The term "nonaqueous carrier" refers to a fluid carrier which is notwater-based, such as acetone or an alcohol such as ethanol.

The term "nonfibrillar collagen" refers to collagens in which the triplehelical molecules do not aggregate to form thick fibers, such that acomposition containing nonfibrillar collagen will be optically clear.

The term "sterile filtration" refers to removal of microbialcontaminants from a solution by passing the solution through a filterhaving pores of a diameter small enough that microbial contaminants willnot pass through the filter pores with the solution that is beingdecontaminated.

The terms "synthetic hydrophilic polymer" or "synthetic polymer" referto polymers which have been synthetically produced and which arehydrophilic, but not necessarily water-soluble. Examples of synthetichydrophilic polymers which can be used in the practice of the presentinvention are polyethylene glycol (PEG), polyoxyethylene, polymethyleneglycol, polytrimethylene gIycols, polyvinylpyrrolidones,polyoxyethylene-polyoxypropylene block polymers and copolymers, andderivatives thereof. Naturally occurring polymers such as proteins,starch, cellulose, heparin, hyaluronic acid, and derivatives thereof areexpressly excluded from the scope of this definition.

The term "syringe" refers to a device which is adapted for the injectionof a material to a tissue site, which may be soft or hard tissue.

The term "tissue augmentation" as used herein refers to the replacementor repair of defects in the soft or hard tissues of a human body.

Except as otherwise defined above, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Although anymethods and materials similar or equivalent to those described hereinmay be useful in the practice or testing of the present invention, onlythe preferred methods and materials are described below. It is notintended that the invention be limited to these preferred embodiments,however. The invention is intended to have the scope defined by theattached claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

In order to practice the methods of the present invention, it is firstnecessary to provide a biocompatible polymer, such as, for example,collagen or a glycosaminoglycan, and a sterile, dry crosslinking agent,such as a synthetic hydrophilic polymer or a carbodiimide.

Collagen, or derivatives thereof, may be used as the biocompatiblepolymer in the methods of the present invention. Lysine residues oncollagen molecules contain primary amino groups capable of reacting withsynthetic hydrophilic polymers; therefore, collagen can be used in itsnatural, purified state and need not be chemically modified in order toform covalently bound conjugates with synthetic hydrophilic polymers.

Collagen from any source may be used in the practice of the invention;for example, collagen may be extracted and purified from human or othermammalian source, or may be recombinantly or otherwise produced.Collagen of any type may be used, including, but not limited to, typesI, II, III, IV, or any combination thereof, although type I is generallypreferred. Atelopeptide collagen is generally preferred overtelopeptide-containing collagen because of its reduced immunogenicity.Collagens that have been previously crosslinked by radiation, heat, orother chemical crosslinking agents such as glutaraldehyde may be used,but are generally not preferred. The collagen should be in apharmaceutically pure form such that it can be incorporated into a humanbody without generating any significant immune response.

Nonfibrillar collagens, including those that have been chemicallymodified, such as succinylated collagen or methylated collagen, may beused in the practice of the invention, but they are not generallypreferred. Fibrillar collagens are generally preferred for use in softtissue augmentation because their ability to form thick, robust fibershas been demonstrated to result in greater persistence in vivo thannonfibrillar collagens. Fibrillar collagen prepared by methods known inthe art or commercially available atelopeptide fibrillar collagencompositions, such as Zyderm® I Collagen (35 mg/ml collagenconcentration) or Zyderm II Collagen (65 mg/ml collagen concentration),are preferred collagens for use in the methods of the present invention.The collagen concentration of a collagen suspension should generally bewithin the range of about 10 mg/ml to about 120 mg/ml, most preferably,in the range of about 30 mg/ml to about 70 mg/ml. The collagenconcentration of commercially available collagen compositions can bedecreased by mixing the collagen composition with an appropriate amountof sterile water or phosphate buffered saline (PBS). Conversely, toincrease the collagen concentration, the collagen composition can beconcentrated by centrifugation, then adjusted to the desired collagenconcentration by mixing with an appropriate amount of sterile water orPBS.

Glycosaminoglycans, such as, for example, hyaluronic acid, chondroitinsulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate,keratosulfate, chitin, chitosan, heparin, and derivatives or mixturesthereof, may be used as the biocompatible polymer in the methods of thepresent invention. Different types of glycosaminoglycans can be mixedtogether, or mixed with collagen, and used in the practice of theinvention. Glycosaminoglycans must generally be modified, such as bydeacetylation or desulfation, in order to provide primary amino groupscapable of binding with functional groups on synthetic hydrophilicpolymers. Methods for chemically modifying glycosaminoglycans in such amanner that they are capable of binding with synthetic hydrophilicpolymers are described in commonly owned, copending U.S. applicationSer. No. 08/146,843, filed Nov. 3, 1993. In general, glycosaminoglycanscan be deacetylated, desulfated, or both, as applicable, by the additionof a strong base, such as sodium hydroxide, to the glycosaminoglycan.The deacetyled and/or desulfated glycosaminoglycan is capable ofcovalently binding with a functionally activated synthetic hydrophilicpolymer.

Any biocompatible crosslinking agent that is available in dry form, orcan be processed to be in dry form while still retaining crosslinkingactivity, can be used in the device and method of the present invention.However, synthetic hydrophilic polymers, such as functionally activatedpolyethylene glycols, are the preferred crosslinking agents, withdifunctionally activated polyethylene glycols being most preferred.Various activated forms of polyethylene glycol are described below.

Specific Forms of Activated Polyethylene Glycol

For use in the present invention, molecules of polyethylene glycol arechemically modified in order to provide functional groups on one or,preferably, two or more sites along the length of the PEG molecule, sothat covalent binding can occur between the PEG and the reactive groupson the biocompatible polymer. Some specific activated forms of PEG areshown structurally below, as are generalized reaction products obtainedby reacting activated forms of PEG with collagen. In Formulas 1-7, theterm COL represents collagen. The term PEG represents polymers havingthe repeating structure (OCH₂ CH₂)_(n).

The first activated PEG is difunctionally activated PEG succinimidylglutarate, referred to herein as (SG-PEG). The structural formula ofthis molecule and the reaction product obtained by reacting it withcollagen are shown in Formula 1. ##STR1##

Another difunctionally activated form of PEG is referred to as PEGsuccinimidyl (S-PEG). The structural formula for this compound and thereaction product obtained by reacting it with collagen is shown inFormula 2. In any general structural formula for the compound, thesubscript 3 is replaced with an "n". In the embodiment shown in Formula1, n=3, in that there are three repeating CH₂ groups on either side ofthe PEG. The structural in Formula 2 results in a conjugate whichincludes an "ether" linkage which is not subject to hydrolysis. This isdistinct from the conjugate shown in Formula 1, wherein an ester linkageis provided. The ester linkage is subject to hydrolysis underphysiological conditions. ##STR2##

Yet another difunctionally activated form of polyethylene glycol,wherein n=2, is shown in Formula 3, as is the conjugate formed byreacting the activated PEG with collagen. ##STR3##

Another preferred embodiment of the invention similar to the compoundsof Formulas 2 and 3 is provided when n=1. The structural formula andresulting collagen-synthetic polymer conjugate are shown in Formula 4.It is noted that this conjugate includes both an ether and a peptidelinkage. These linkages are stable under physiological conditions.##STR4##

Yet another difunctionally activated form of PEG is provided when n=0.This compound is referred to as PEG succinimidyl carbonate (SC-PEG). Thestructural formula of this compound and the conjugate formed by reactingSC-PEG with collagen is shown in Formula 5. ##STR5##

All of the activated polyethylene glycol derivatives depicted inFormulas 1-5 involve the inclusion of the succinimidyl group. However,different activating groups can be attached at sites along the length ofthe PEG molecule. For example, PEG can be derivatized to formdifunctionally activated PEG propion aldehyde (A-PEG), which is shown inFormula 6, as is the conjugate formed by the reaction of A-PEG withcollagen. The linkage shown in Formula 6 is referred to as a --(CH₂)_(n)--NH-- linkage, where n=1-10. ##STR6##

Yet another form of activated polyethylene glycol is difunctionallyactivated PEG glycidyl ether (E-PEG), which is shown in Formula 7, as isthe conjugate formed by reacting such with collagen. ##STR7##

Many of the activated forms of polyethylene glycol described above arenow available commercially from Shearwater Polymers, Huntsville, Ala.,and Union Carbide, South Charleston, W. Va. The various activated formsof polyethylene glycol and various linkages which can be used to producecollagen-synthetic polymer conjugates having a range of physical andchemical properties are described in further detail in commonly ownedU.S. Pat. No. 5,328,955, issued Jul. 12, 1994.

The concentration of crosslinking agent used in the practice of theinvention will vary depending on the type and concentration ofbiocompatible polymer used, the type of crosslinking agent used, themolecular weight of the crosslinking agent, and the degree ofcrosslinking desired. For example, when reacting a suspension ofcollagen (which has a molecular weight of approximately 300,000) havinga collagen concentration of approximately 35 mg/ml with a difunctionallyactivated SG-PEG having a molecular weight of approximately 3800, theconcentration of SG-PEG used is generally within the range of about 1milligram to about 20 milligrams of difunctionally activated SG-PEG(DSG-PEG) per milliliter of collagen suspension, representing a molarratio of between about 2 to about 48 moles of DSG-PEG per mole ofcollagen. When using a suspension of collagen having a collagenconcentration of approximately 65 mg/ml, the concentration ofdifunctionally activated SG-PEG used is generally within the range ofabout 2 milligrams to about 40 milligrams of SG-PEG per milliliter ofcollagen suspension.

Preparation of a Sterile, Dry. Crosslinking Agent Contained Within aSyringe

Various methods can be used to prepare sterile, dry crosslinking agentscontained within syringes for use in the methods of the presentinvention. Preferred methods are described below.

In Method A, a dry crosslinking agent is dissolved in a fluid carrier,which may be an aqueous carrier or a nonaqueous carrier. Suitablenonaqueous carriers include ethanol, methylene chloride, acetone, orchloroform, with ethanol being particularly preferred. If an aqueouscarrier is used in conjunction with a water-reactive crosslinking agent,such as a functionally activated polyethylene glycol, the carrier shouldbe maintained at a slightly acidic pH, preferably, between about pH 3and about pH 5, in order to retard hydrolysis of the crosslinking agent.After the crosslinking agent has been dissolved in the fluid carrier,the resulting crosslinker solution is sterile-filtered through one ormore filters having pore sizes of 0.22 microns or smaller to produce asterile crosslinker solution. The sterile crosslinker solution is thendispensed into syringes (preferably, about 3 cc in volume), each ofwhich may optionally be fitted with a sterile barrier cap, such as a 0.2micron hydrophobic polytetrafluoroethylene (PTFE) membrane. The sterilecrosslinker is then lyophilized aseptically within the syringe toproduce a sterile, dry crosslinking agent.

In a method similar to Method A, above, a dry crosslinking agent isdissolved in a fluid carrier, which may be an aqueous carrier or anonaqueous carrier. After the crosslinking agent has been dissolved inthe fluid carrier, the resulting crosslinker solution is then dispensedinto syringes, each of which may optionally be fitted with a sterilebarrier cap, and lyophilized within the syringe to produce a drycrosslinking agent. The dry crosslinking agent is then sterilized insidethe syringe using irradiation, which is preferably e-beam or gammairradation.

In an alternative method, Method B, a dry crosslinking agent isdissolved in a nonaqueous carrier. The resulting nonaqueous crosslinkersolution is then sterile-filtered through one or more filters havingpore sizes of 0.22 microns to produce a sterile crosslinker solution.The sterile crosslinker solution is then dispensed into syringes anddried inside the syringes by evaporation under vacuum at a temperaturein the range of about 10° C. to about 20° C. under aseptic conditions.When a nonaqueous carrier is used with a water-reactive crosslinkingagent, there is no need to worry about loss of crosslinking activity dueto hydrolysis.

In a method similar to Method B, above, a dry crosslinking agent isdissolved in a nonaqueous carrier. The resulting nonaqueous crosslinkersolution is then dispensed into syringes and dried by evaporation undervacuum at a temperature in the range of about 10° C. to about 20° C. Thedry crosslinking agent is then sterilized inside the syringes usingirradiation, which is preferably e-beam or gamma irradiation.

In yet another method, Method C, a dry crosslinking agent is firstdispensed into syringes, then sterilized inside the syringes usingirradiation, preferably e-beam or gamma irradiation. Irradiation of thedry crosslinking agent within the syringe is preferably performed in theabsence of oxygen to minimize oxidation of the crosslinking agent duringthe irradiation process.

To facilitate dispensing of the crosslinking agent into syringes, thedry crosslinking agent may be compressed into a mold to form a pellet.The crosslinking agent may be molded into pellets neat (withoutadditives) or mixed (prior to molding) with a filler material such as abiocompatible dry inert agent in order to increase the volume ofcrosslinking agent to be dispensed into each syringe. Suitable dry inertagents include unactivated polyethylene glycols, sugars, salts, orcarbohydrates. Preferred inert agents include glucose and sodiumchloride. Dry inert agents are generally added to the dry crosslinkingagent in a ratio of between about 5 to about 50 milligrams of dry inertagent per milligram of crosslinking agent and, preferably, between about20 to about 30 milligrams of inert agent per milligram of crosslinkingagent.

In a particularly preferred embodiment of Method C, the dry crosslinkingagent is molded to form a pellet and sterilized using e-beamirradiation. Preferably, the pellet is dispensed into a syringe prior toirradiation; however, it is possible to sterilize the pellet beforeplacing it in the syringe.

We initially had some concern regarding the effectiveness of e-beamsterilization, as the e-beam radiation does not penetrate as deeply asother types of radiation, such as gamma irradiation. However, ourexperiments indicate that e-beam radiation results in sterility levelswell within the range of acceptability for a product intended forincorporation into the body of a human patient. The e-beam irradiationis preferably within the range of about 0.5 Mrad to about 4 Mrad; mostpreferably, between about 1.5 Mrad to about 2.5 Mrad.

Surprisingly, our experiments have shown that dry crosslinking agentsshow better retention of crosslinking ability when sterilized usinge-beam irradiation than when gamma irradiation is used. Pelletssterilized using gamma irradiation at dosage levels higher than about1.5 Mrad showed a significant loss of crosslinking activity; a totalloss of crosslinking activity was seen at 2.5 Mrad using gammairradiation. Pellets sterilized using e-beam irradiation up to a dosagelevel of 4 Mrad showed no loss of activity in comparison to anon-irradiated control pellet. Depending on the desired end use of thedry crosslinking agent, gamma irradiation at higher dosage levels mayresult in an unacceptable loss of crosslinking activity, whereas e-beamirradiation at dosage levels up to at least 4 Mrad appears to have noeffect on the activity of the crosslinking agent and is capable ofachieving sterility levels well within the acceptable range for aninjectable or implantable product.

Method of Preparing Crosslinked Biomaterial Compositions

In order to practice the method of the present invention, it is firstnecessary to provide a means for delivering a biocompatible polymer,such as collagen or a glycosaminoglycan derivative, and a means fordelivering a sterile, dry crosslinking agent, such as a synthetichydrophilic polymer. Syringes are the presently preferred means fordelivering the biocompatible polymer and the crosslinking agent,although other devices, such as compressed air injectors, may beutilized. We have discovered that optimum crosslinking is achieved whenthe syringe containing the biomaterial or the syringe containing the drycrosslinking agent (or both syringes) has a contraction ratio (i.e., theratio of the inner diameter of the barrel to the inner diameter of thesyringe exit orifice) of about 4.0 or greater. Preferred syringes foruse in the present invention have a barrel volume of about 3 cc, abarrel diameter of about 8.5 mm, an exit orifice diameter of about 2 mm,and a contraction ratio of about 4.25. To achieve optimum mixing, thepreferred volume of biomaterial contained within the syringe is betweenabout 1 cc and about 2 cc. Non-uniform mixing could lead to the creationof regions of low crosslink density within an implant, which couldsignificantly weaken it, leading to poor persistence. Poor mixing couldalso create regions of high local crosslinker concentration within theimplant; the excess crosslinker could leach into tissues surrounding theimplant, rather than serving to crosslink the implant or to anchor theimplant to adjacent tissue.

In a preferred method for preparing crosslinked biomaterialcompositions, a syringe containing a biomaterial and a syringecontaining a sterile, dry crosslinking agent are provided. The syringecontaining the biomaterial and the syringe containing the drycrosslinking agent are then connected by means of a syringe connector.Once the connection between the two syringes has been established, thebiomaterial and the dry crosslinking agent are mixed back and forthbetween the syringes. When the biomaterial and crosslinking agent havebeen adequately mixed, all of the material is transferred into one ofthe two original syringes, or into a third syringe. The material canthen be extruded from the syringe orifice into molds of the desired sizeand shape to produce a variety of formed implants, including tubularimplants for use as vascular grafts or stents, or it can be extrudedonto one or more surface of a preformed synthetic implant, such as abone prosthesis or synthetic vascular graft or stent, to provide acrosslinked, nonimmunogenic biomaterial coating on the surface of theimplant. Alternatively, a needle can be attached to the syringe fromwhich the material can be extruded to provide soft tissue augmentationin a patient.

The material must be extruded from the syringe before completecrosslinking has occurred between the biocompatible polymer and thecrosslinking agent. The amount of time required for completecrosslinking to be achieved depends on a variety of factors, includingthe type and concentration of biocompatible polymer used, the type andconcentration of crosslinking agent used, and the temperature and pH ofthe materials. For example, when a collagen suspension having a collagenconcentration of approximately 35 mg/ml and a pH of approximately 7 isreacted with a difunctionally activated SG-PEG (DSG-PEG, 3800 MW) at aconcentration of about 1-20 milligrams DSG-PEG per milliliter ofcollagen suspension, complete crosslinking between the collagen and theDSG-PEG at room temperature is generally achieved within 20 to 30minutes of the initiation of mixing between the two components. Thecrosslinking reaction will generally proceed more slowly if the reactionis performed at a lower temperature and/or lower pH.

In a preferred method for providing soft tissue augmentation, aphysician is provided with a kit comprising the following: one 3-ccsyringe containing a biocompatible polymer, which is preferablycollagen; one 3-cc syringe containing a sterile, dry crosslinking agent,which is preferably a synthetic hydrophilic polymer such as afunctionally activated polyethylene glycol; one empty 1-cc syringe; onesyringe connector (such as a three-way stopcock); and one or moreneedles, which are preferably 25-gauge or smaller, more preferably,27-gauge or smaller and, most preferably, 30-gauge. The size of theneedles provided will depend on the intended site of application of thecrosslinked biomaterial. For example, for hard tissue applications orsoft tissue applications such as sphincter augmentation, a 25-gauge orsmaller needle is acceptable. However, for soft tissue applications suchas dermal contour correction in the face, a 27-gauge or smaller needleis required, with a 30-gauge needle being most preferred.

The physician connects the syringe containing the biomaterial with thesyringe containing the dry crosslinking agent by means of the syringeconnector. He/she then mixes the biomaterial and crosslinking agent backand forth between the syringes, preferably employing at least 20, and,preferably, at least 30, passes of material between the syringes (onepass is counted each time the volume of material passes through thesyringe connector). When the biomaterial and crosslinking agent havebeen adequately mixed, the physician transfers all of the material intoone of the 3-cc syringes, detaches the empty 3-cc syringe from thesyringe connector, attaches the empty 1-cc syringe to the 3-cc syringecontaining biomaterial and crosslinking agent by means of the syringeconnector, then transfers the entire contents of the 3-cc syringe intothe formerly empty 1-cc syringe. He/she then attaches one of the needlesto the full 1-cc syringe and injects the biomaterial and crosslinkingagent, which have since initiated crosslinking, to the tissue site inneed of augmentation. Preferably, treatment of the patient is completedwithin 20 to 30 minutes of mixing the biomaterial and the crosslinkingagent.

In a less preferred embodiment, the biocompatible polymer and thesterile, dry crosslinking agent are contained in separate barrels of adouble-barreled syringe which has a chamber or compartment housing ameans for mixing the polymer and the crosslinking agent in advance ofthe syringe orifice. When the physician exerts pressure on the syringeplunger, the biocompatible polymer and dry crosslinking agent are bothextruded into the mixing chamber, where mixing and some preliminarycrosslinking of the biocompatible polymer with the crosslinking agentoccur prior to their extrusion from the needle to the intended tissuesite, where further crosslinking takes place.

In another, less preferred embodiment, a lyophilized biocompatiblepolymer, a sterile, dry crosslinking agent, and a biocompatible fluidcarrier are all contained in the barrel of one syringe, with the threecomponents being separated one from the other by means of sterilebarriers. When depressed by the physician, the syringe plunger piercesthe sterile barriers, allowing the three components--biocompatiblepolymer, crosslinking agent, and fluid carrier--to come in contact withone another, rehydrate, and mix prior to being delivered from thesyringe needle to the tissue site in need of augmentation.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake the preferred embodiments of the conjugates, compositions, anddevices and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, molecularweight, etc.) but some experimental errors and deviation should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1

(Mixing Efficiency of Dry and Aqueous Crosslinkers With CollagenSuspensions)

The following experiment was performed to evaluate the mixing efficiencyof difunctionally activated SG-PEG (DSG-PEG, 3800 MW) and collagen underseveral different mixing protocols, as set forth in Table 1. Variablesexamined included syringe size, number of mixing passes, and whether theDSG-PEG was in aqueous solution (liquid) or dry (powder) form.

                  TABLE 1                                                         ______________________________________                                        Mixing Protocols for Collagen/DSG-PEG Formulations                            DSG-PEG   Collagen             # of                                           Syringe   Syringe  Form of     Mixing                                                                              Sample                                   Size (cc) Size (cc)                                                                              DSG-PEG     Passes                                                                              Code                                     ______________________________________                                        1         1        Liquid      20    1:1L20                                   1         1        Liquid      40    1:1L40                                   1         3        Liquid      20    1:3L20                                   1         3        Powder      20    1:3P20                                   1         3        Liquid      40    1:3L40                                   1         3        Powder      40    1:3P40                                   3         3        Liquid      20    3:3L20                                   3         3        Powder      20    3:3P20                                   3         3        Liquid      40    3:3L40                                   3         3        Powder      40    3:3P40                                   ______________________________________                                    

Syringes having a barrel volume of 1-cc (Part No. 4174, CIMCO MedicalCorporation, Costa Mesa, Calif.) had an inner barrel diameter of 5.5 mm,a barrel length of 70 mm, and an orifice inner diameter of 2.5 mm.Syringes having a barrel volume of 3-cc (Part No. 309585, BectonDickinson Corporation, Franklin Lakes, N.J.) had an inner barreldiameter of 8.5 mm, a barrel length of 60 mm, and an orifice innerdiameter of 1.8 mm.

Samples involving aqueous solutions of DSG-PEG were prepared accordingto the following procedure: 0.9 gram of Zyderm® I Collagen was weighedinto a 1-cc syringe (samples 1:1L20 and 1:1L40) or into a 3-cc syringe(samples 1:3L20, 1:3L40, 3:3L20, and 3:3L40). Ten (10) milligrams of dryDSG-PEG was weighed into a tared Eppendorf tube. Two hundred (200)microliters of 0.02M phosphate-buffered saline (PBS, pH=7.3) was thenpipetted into the tube. The sample was vortexed for 1 minute, then 100milliliters of the sample was pipetted into the barrel of a 1-cc syringe(samples 1:1L20, 1:1L40, 1:3L20, 1:3L40) or a 3-cc syringe (samples3:3L20 and 3:3L40). The DSG-PEG solution was compressed to the tip ofthe syringe using the syringe plunger. Care was taken to expel alltrapped air, yet retain all of the crosslinker solution. The collagen inits syringe was also compressed to the tip, so as to expel all trappedair, yet retain all of the collagen.

Samples involving dry DSG-PEG were prepared according to the followingprocedure: One (1.0) gram of Zyderm® I Collagen was weighed into a 3-ccsyringe. Five (5) milligrams of DS G-PEG powder was weighed into eithera 1-cc syringe (samples 1:3P20) and 1:3P40) or a 3-cc syringe (samples3:3P20 and 3:3P40). The DSG-PEG powder was compressed to the tip of thesyringe using its plunger. Care was taken to expel all air from thesyringe, yet retain all of the SG-PEG powder. The collagen in itssyringe was compressed to the tip so as to expel all trapped air, yetretain all of the collagen.

All samples were mixed according to the following procedure: A three-waystopcock (Catalog No. K75, Baxter Healthcare Corporation, PharmaSealDivision, Valencia, Calif.) was connected to the syringe containing thecollagen. The collagen was extruded through the connector orifice sothat there would be no trapped air when the syringe containing theDSG-PEG was connected, but also so that no collagen was lost. Thesyringe containing the DSG-PEG (in solution or dry) was connected to thestopcock-collagen syringe assembly. Mixing of the DSG-PEG and thecollagen was performed by simple exchange between syringes. One pass wascounted each time the volume of the DSG-PEG--collagen mixture passedthrough the stopcock. Plunger strokes were made as quickly as possible.In general, all of the mixing (from first pass to last) was accomplishedwithin 1 minute. After mixing was completed and the entire volume ofmixture was contained in one syringe, the empty mixing syringe wasremoved and replaced with a clean 1-cc syringe. The DSG-PEG--collagenmixture was loaded by compression into the clean 1 -cc syringe, whichwas then capped and placed in a 37° C. incubator to crosslink overnight,forming a gel.

The tip end of the 1 -cc syringe was cut off and the plunger used togently eject the cylindrical gel from the syringe. The gel wassubsequently sectioned into 4-mm thick disks for mechanical strengthtesting. Samples were compressed to failure in the Instron UniversalTester, Model 4202, at a constant rate of 2 millimeters per minute.Results of compression testing are presented in Table 2. Sample positionis listed starting from the tip to the plunger end of the syringe.Samples that had air bubbles or that were too thick are not included inthe results.

                                      TABLE 2                                     __________________________________________________________________________    Compression Testing of Collagen - DSG-PEG Gels                                Force to Failure (in Newtons)                                                 Sample Code                                                                   Sample                                                                             1:1                                                                              1:1 1:3                                                                              1:3 1:3                                                                              1:3 3:3                                                                              3:3 3:3                                                                              3:3                                       Position                                                                           L20                                                                              L40 L20                                                                              P20 L40                                                                              P40 L20                                                                              P20 L40                                                                              P40                                       __________________________________________________________________________    Tip  7.6                                                                              5.5 13.9                                                                             12.3                                                                              9.8                                                                              8.3 13.5                                                                             8.5 11.6                                                                             12.5                                      Tip + 1                                                                            5.6                                                                              7.8 16.1                                                                             8.3 7.9                                                                              8.0 14.6                                                                             12.4                                                                              9.3                                                                              14.5                                      Tip + 2                                                                            n/a                                                                              10.5                                                                              12.1                                                                             8.4 11.1                                                                             8.2 10.3                                                                             10.7                                                                              11.6                                                                             10.8                                      Tip + 3                                                                            6.1                                                                              11.6                                                                              9.2                                                                              9.2 9.1                                                                              8.0 11.4                                                                             10.8                                                                              10.9                                                                             10.8                                      Tip + 4                                                                            9.0                                                                              9.8 5.0                                                                              n/a 9.8                                                                              9.2 12.3                                                                             11.9                                                                              n/a                                                                              9.0                                       Tip + 5                                                                            7.1                                                                              10.3                                                                              8.6                                                                              9.8 9.1                                                                              10.4                                                                              n/a                                                                              n/a 8.4                                                                              n/a                                       Tip + 6                                                                            n/a                                                                              10.8                                                                              8.7                                                                              7.9 8.4                                                                              10.4                                                                              n/a                                                                              n/a 7.7                                                                              n/a                                       Tip + 7                                                                            n/a                                                                              n/a 5.5                                                                              n/a n/a                                                                              9.2 n/a                                                                              n/a n/a                                                                              n/a                                       Plunger                                                                            11.4                                                                             10.1                                                                              3.3                                                                              9.7 10.6                                                                             8.2 12.5                                                                             n/a 11.7                                                                             n/a                                       N =  6  8   9  7   8  9   6  5   7  5                                         Mean 7.8                                                                              9.6 9.2                                                                              9.4 9.5                                                                              8.9 12.4                                                                             10.9                                                                              10.2                                                                             11.5                                      Range*                                                                             5.8                                                                              6.1 12.8                                                                             4.4 3.2                                                                              2.4 4.3                                                                              3.9 3.3                                                                              5.5                                       __________________________________________________________________________     *Range = highest value minus lowest value.                               

Failure force measurements are heavily influenced by the presence ofphysical defects in the gel samples. The purpose of applying this typeof measurement was to detect defects resulting from poor mixing ofcollagen and crosslinker. Mixing defects consist of regions of lowcrosslinker concentration, as well as the creation of stress planes bydeforming the gel as network formation was occurring, essentially actingto fragment the gel. It is important to form strong, uniformlycrosslinked gels because, when implanted in vivo, weaker gels tend to bedisplaced and deformed more over time by pressures exerted by thesurrounding tissue than would strong gels.

Variability in gel strength measurements was examined by preparing fourgels identical to sample 3:3P40. The gels were allowed to cure for 6hours at 37° C. and then were sectioned into 4-mm thick disks formechanical testing. Results of mechanical compression testing arepresented in FIG. 1. The results show that, with the exception of onemeasurement, all samples possessed failure strengths greater than 10Newtons, with a scatter about the mean of up to 2 Newtons (i.e., a rangeof about 4 Newtons). There is insignificant variability in gel strengthfrom the tip of the syringe to the plunger end. This material maytherefore be concluded to have good mixing.

Based on the results of our experiments, two interpretation guidelinescan be drawn: 1) disks with gel strengths of approximately 6 Newtons orless (representing more than two times the scatter that could beexpected from preparation variability) can be considered weak; weak gelsare indicative of areas of low local crosslinker concentration resultingfrom poor mixing; 2) trends in disk strength or variations in diskstrength beyond the expected 2 Newton scatter suggest poor mixing of thecollagen and crosslinker.

Based on the above interpretation guidelines, the gels prepared bymixing between 1-cc syringes displayed trends (sample 1:1L40) orvariability (1:1L20) strongly suggestive of poor mixing. There areseveral sections within these two gels (especially for sample 1:1L20)that are very weak. Gels mixed for 40 passes showed significantimprovement in mechanical properties as compared to the gels mixed foronly 20 passes.

Mixing between 1- and 3-cc syringes using DSG-PEG in aqueous solutionshows a similar improvement as the number of passes are increased. Thegels mixed for 20 passes clearly show a trend in strength according togel position and there are weak sections in the gel, indicatinginsufficient mixing. Forty passes appears to give reasonable mixing anduniform strength in a range indicative of reasonable crosslinkdensities.

Mixing between 1- and 3-cc syringes using DSG-PEG powder appears to giveadequate mixing. However, the strengths of several of the disks are onthe low end (approximately 8 Newtons) of the expected variability range.

Mixing between 3-cc syringes, with either aqueous or powder forms ofDSG-PEG, gave uniformly strong gel sections within the variabilityrange. There appears to be one weak spot in sample 3:3L40; given theaverage higher strength of 3:3L20, which had less mixing time, this islikely a result of stress plane generation resulting from continuedmixing of the sample after gel formation had been initiated.

Example 2 (Preparation of a Sterile, Dry Crosslinking Agent:

Lyophilization of Difunctionally Activated SG-PEG)

Fifteen hundred (1500) milligrams of difunctionally activated SG-PEG(DSG-PEG, 3800 MW) was mixed with 150 milliliters of water-for-injection(WFI, pH 4.5), resulting in a final DSG-PEG concentration ofapproximately 10 mg/ml. The DSG-PEG solution was then sterile-filteredthrough a Durapore filter having a pore size of 0.22 microns (MilliporeCorporation, Bedford, Mass.). 0.5 milliliter of DSG-PEG solution wasaliquotted into each of 180 3-cc syringes. Each syringe was then fittedwith a sterile barrier cap comprising a 0.2 micron hydrophobicpolytetrafluoroethylene membrane (Part No. 187-1320, Nalgene Company,Rochester, N.Y.). The syringes were placed in racks and lyophilizedusing the following lyophilization cycle in the lyophilizer (Model 15SRC-X, Virtis Company, Gardiner, N.Y.).

    ______________________________________                                               Set Point                                                                            Ramp Time  Soak Time Vacuum                                     ______________________________________                                        Segment 1                                                                              -40° C.                                                                         1.0 hr     5.0 hr  OFF                                      Segment 2                                                                               -5° C.                                                                         3.0 hr     3.0 hr  ON                                       Segment 3                                                                              +20° C.                                                                         2.0 hr     12.0 hr ON                                       ______________________________________                                    

Example 3 (HPLC Analysis of Lyophilized DSG-PEG)

The following experiment was performed to measure the activity oflyophilized DSG-PEG to determine if lyophilization had resulted in anunacceptable loss of activity of the DSG-PEG:

The contents of syringes prepared according to the procedure describedin Example 2 were analyzed by HPLC for DSG-PEG content and compared toan aqueous control sample of DSG-PEG which had been solubilized andsterile-filtered. Lyophilized samples were analyzed for DSG-PEG contentimmediately after lyophilization and again after 2-week storage with adesiccant at room temperature. The aqueous control sample of DSG-PEG wasanalyzed approximately 2 hours after solubilization. HPLC analysis ofDSG-PEG samples was performed using an isocratic elution. Conditions ofHPLC analysis were as follows:

Column: Waters Ultrahydrogel 250

Pore Size: 250 Angstroms

Column Size: 7.8 mm×30 cm

Exclusion Limit: 8×10⁴ daltons

Injection Volume: 20 μl

Mobile Phase: 5 mM Sodium Acetate buffer, pH=5.5 at 21° C.

Flow Rate: 0.5 ml/min

Pressure: 0.8 mPa

Detector: Dual Detector System, Refractive Index & UV at 260 nm

An external standard calibration curve was obtained using PEG solutionsof various concentrations. The stock solution was prepared by dissolving10.0 mg of difunctionally activated SG-PEG in 1.000 ml of deionizedwater. The solution was sequentially diluted to 5.00, 2.50, 1.25, 0.625,and 0.3125 mg/ml and analyzed by HPLC. Integrating the peak at aretention time of 16 minutes, the peak area was plotted against eachconcentration of DSG-PEG standard.

The aqueous control sample of DSG-PEG showed 76.3% retention ofdifunctionality, as compared to 74.1% for the sample tested immediatelyfollowing lyophilization, and 73.9% (average of two samples) for samplestested after 2-week storage with desiccant at room temperature.Difunctionality is a measure of the potential ability of a substance tocrosslink two or more molecules of collagen and/or other biocompatiblepolymer (i.e., "monofunctional" polymer molecules are not capable ofperforming crosslinking because they contain only one functional groupand are therefore capable of reacting with only one molecule ofbiocompatible polymer.)

Example 4 (Stability of Previously Frozen SG-PEG Solutions)

The following experiment was performed to determine if solutions ofdifunctionally activated SG-PEG in water-for-injection would retaintheir activity when frozen for periods of time as long as 3 weeks inorder to determine if DSG-PEG solutions could be pre-frozen, thenlyophilized in large batches.

A solution having a concentration of 10 milligrams of DSG-PEG permilliliter of WFI (pH 4.5) was prepared. The DS G-PEG solution wasaliquotted, 1 ml each, into Eppendorf tubes. A control sample wasremoved for HPLC analysis. The remainder of the samples were placed in afreezer at -20° C. Samples were removed from the freezer at various timeintervals, allowed to thaw at room temperature for 10 minutes, thenanalyzed for DSG-PEG content by HPLC according to the procedure detailedin Example 3. Results of HPLC analysis are presented in Table 3.

                  TABLE 3                                                         ______________________________________                                        HPLC Analysis of Previously Frozen DSG-PEG in WFI                             Time           % DSG-PEG                                                      ______________________________________                                        0         (Control)                                                                              86.3                                                       2         hours    84.6                                                       1         day      82.7                                                       2         days     82.4                                                       3         days     80.7                                                       6         days     83.5                                                       8         days     82.2                                                       22        days     78.6                                                       ______________________________________                                    

Results show that DSG-PEG in WFI retains at least 90% of its originalactivity upon freezing for up to 22 days at -20° C.

Example 5 (Preparation of a Dry Crosslinking Agent: Pelletization ofDSG-PEG)

Difunctionally activated SG-PEG (DSG-PEG, 3800 MW) was pelleted withglucose, unactivated polyethylene glycol (PEG), and/or sodium chloride.The dry reagents were mixed using a mortar and pestle, then loaded intoa compression mold to form pellets. The following four formulations wereprepared:

Formulation 1: 7.5 mg DSG-PEG, 30.0 mg glucose, 7.5 mg PEG(DSG-PEG/Glucose/PEG)

Formulation 2: 7.5 mg DSG-PEG, 37.5 mg glucose (DSG-PEG/Glucose)

Formulation 3: 7.5 mg DSG-PEG, 75.0 mg NaCl (DSG-PEG/NaCl)

Formulation 4: 7.5 mg DSG-PEG, 30.0 mg NaCl 7.5 mg PEG(DSG-PEG/NaCl/PEG)

Each of the fore pellets was placed in the barrel of a 3.0-cc syringeand mixed with 1.5 cc of Zyderm® I Collagen (35 mg/ml collagenconcentration, available from Collagen Corporation, Palo Alto, Calif.)in a 3.0-cc syringe. The pellets quickly solubilized when contacted withthe collagen. The collagen and pellets were mixed for 2 minutes usingsyringe-to-syringe mixing, as previously described. All of the materialfor each formulation was transferred into one syringe, then allowed tocrosslink within the syringe for 3 hours. Each of the four formulationshad a molar ratio of approximately 12 moles of DSG-PEG per mole ofcollagen.

Example 6 (Gel Strength Testing of Collagen Crosslinked with PelletizedDSG-PEG)

The four cylindrical gels of PEG-crosslinked collagen prepared inExample 5 were pushed out of their respective syringes, then sliced intodisks having a thickness of 5 mm each. Gel strength of each of thePEG-collagen disks was measured by compressing the disks to failureusing the Instron Model 4202 Universal Testing Instrument with 100Newton Static Load Cell. Gel strength for each of the four formulationsis presented in Table 4.

                  TABLE 4                                                         ______________________________________                                        Gel Strength Measurements for PEG-Collagen Formulations                       Gel Strength (in Newtons)                                                     Crosslinking                                                                           DSG-PEG/                                                             Agent    Glucose/  DSG-PEG/  DSG-PEG/                                                                              DSG-PEG/                                 Composition:                                                                           PEG       Glucose   NaCl    NaCl/PEG                                 ______________________________________                                        Sample 1 39.4      28.3      34.6    32.7                                     Sample 2 24.9      29.1      38.0    30.1                                     Sample 3 27.0      25.0      39.4    37.9                                     Sample 4 35.7      27.1      n/a     39.7                                     Average  31.8      27.4      37.3    35.1                                     ______________________________________                                    

All four gels showed good gel strength, particularly the gels containingsodium chloride, which may be due to partial disassembly of the collagenfibers by sodium chloride resulting in more sites being available on thecollagen for crosslinking with difunctionally activated SG-PEG. Asnon-crosslinked collagen has no gel strength (i.e., gel strength=0Newtons), the data presented in Table 1 indicate that crosslinking hasoccurred between collagen and DSG-PEG using dry DSG-PEG, with or withoutglucose or sodium chloride.

Factors such as pH, which may vary in vivo depending on the site ofinjection, may affect the rate of the crosslinking reaction between thecollagen and the crosslinking agent, as well as the gel strength of theresulting crosslinked collagen implant.

Example 7 (Method of Preparing and Administering Crosslinked CollagenCompositions for Soft Tissue Augmentation)

A physician opens a kit containing the following components: one 3-ccsyringe containing 1 ml of Zyderm® I Collagen; one 3-cc syringecontaining 10 mg of sterile, dry, difunctionally activated SG-PEG(DSG-PEG, MW 3800); one empty 1-cc syringe; one three-way stopcock; andseveral 30-gauge needles. The physician connects the syringe containingthe Zyderm Collagen with the syringe containing the dry DSG-PEG by meansof the three-way stopcock. She then mixes the collagen and DSG-PEG backand forth between the syringes, employing 40 passes of material betweenthe syringes. She then transfers all of the material into one of the3-cc syringes, detaches the empty 3-cc syringe from the stopcock,attaches the empty 1-cc syringe to the 3-cc syringe containing thecollagen and DSG-PEG by means of the stopcock, then transfers the entirecontents of the 3-cc syringe into the formerly empty 1-cc syringe. Shethen attaches one of the 30-gauge needles to the full 1-cc syringe.

After attaching the needle to the syringe, the physician injects thecollagen and DSG-PEG (which have since initiated crosslinking)subcutaneously, using multiple serial punctures, to soft tissue sites inneed of augmentation, such as acne scars and wrinkles. Treatment of thepatient is completed within 20 minutes of mixing the collagen and theDSG-PEG.

Example 8 (Preparation of a Sterile, Dry Crosslinking Agent:Pelletization of DSG-PEG Followed By E-Beam Irradiation)

Dry, difunctionally activated SG-PEG (DSG-PEG, 3800 MW) was heatedinside a glass syringe for the purpose of facilitating extrusion of thematerial from the syringe. The DSG-PEG was extruded in string form ontoa stainless steel plate, then the string was cut into pieces. The pieceswere weighed and pieces having a weight of 2.5±0.5 mg were selected forfurther testing.

The cut pieces were placed inside 3-cc syringes, one piece per syringe.The syringes were placed into a stopper placement unit, then purged withnitrogen and sealed under vacuum. The sealed syringes were placed intofoil pouches and the pouches vacuum sealed. The sealed pouchescontaining the syringes containing the dry DSG-PEG were e-beamirradiated at 2.5 Mrad and stored in a controlled environment atapproximately 4° C.

Samples from this lot were tested for sterility and endotoxin and passedboth tests. The endotoxin n result was less than 0.5 Eu/ml. The productwas put on stability and retained a gel strength of over 21 Newtonsafter 6 months. Unfortunately, the product exhibited a loss ofdifunctional purity during the 6 months, which may be due to heating thematerial prior to extrusion from the syringe, exposure to moistureduring processing, and/or omission of desiccant in the foil pouch.

The method used to establish the feasibility of using e-beam tosterilize product in 3-cc syringes is based on the AAMI's "Guideline forE-beam Radiation Sterilization of Medical Devices" (Association for theAdvancement of Medical Instrumentation, 1991 ). A known quantity ofspores of B. pumilis were dispersed in glucose powder and filled into3-cc syringes. The filled syringes were irradiated using e-beam atapproximately 2.5 Mrad and the contents tested for sterility. Asterility assurance level of 1×10⁻³ was achieved. The samples processedby the method described above passed sterility tests to achieve asterility assurance level (SAL) of 1×10⁻⁶.

Example 9 (A Comparison of the Effects of Gamma and E-Beam Irradiationon Difunctional Activity of DSG-PEG)

We evaluated the effects of two methods of sterilization--gamma ande-beam irradiation--on difunctional activity of DSG-PEG. Difunctionallyactivated SG-PEG was irradiated using varying doses of either gamma ore-beam irradiation. The irradiated SG-PEG was analyzed for difunctionalpurity by HPLC according to the method described in Example 3, above.Results are presented in Table 5, below.

                  TABLE 5                                                         ______________________________________                                        Sterilization of Difunctionally Activated SG-PEG                              Using Gamma or E-beam Irradiation                                             Radiation  % Difunctional Purity as Measured by HPLC                          Dosage (Mrad)                                                                            Gamma Irradiation                                                                            E-beam Irradiation                                  ______________________________________                                        Control    79.8           88.6                                                0.5        80.5           --                                                  1.0        75.6           --                                                  1.5        75.9           --                                                  2.0        --             85.1                                                2.5        0              87.1                                                ______________________________________                                    

The data indicate that the DSG-PEG could be irradiated using gammairradiation at low dosages, but there was a significant loss ofdifunctionality at gamma irradiation dosages greater than about 1.5Mrad.

Additional experimentation demonstrated that DSG-PEG experienced nosignificant loss of difunctional activity at e-beam irradiation levelsas high as 4.0 Mrad. E-beam irradiation was therefore chosen as thepreferred method of sterilization.

Example 10 (Preparation of a Sterile, Dry Crosslinking Agent:Pelletization of DSG-PEG Followed By E-Beam Irradiation)

An alternative method for preparing the sterile packaged DSG-PEG wasconducted: Dry DSG-PEG was compressed and formed into pellets having aweight of 2.5±0.5 mg. The pellets were placed, one pellet per syringe,into 3-cc plastic syringes. The filled syringes were placed into astopper placement unit. The syringes were sealed under vacuum, thenplaced inside foil pouches containing desiccant and vacuum-sealed.

The sealed pouches were then e-beam sterilized at 1.5 Mrad and stored ina controlled environment at approximately 4° C. for over 4 months.Samples were evaluated for difunctional activity using the HPLC methoddescribed in Example 3, above, and compared to a non-irradiated control.The control sample exhibited 91% difunctional activity. The packaged,irradiated samples kept in storage at 4° C. exhibited greater than 88%difunctional activity. It can be concluded that the packaging andterminal sterilization process described above is capable of producing asterile, crosslinking agent with no significant loss of activity.

What is claimed is:
 1. A process for preparing a sterile, drycrosslinking agent comprising molding a dry crosslinking agent to form apellet and sterilizing the pellet using e-beam irradiation, wherein thecrosslinking agent is a functionally activated polyethylene glycol. 2.The process of claim 1, wherein the functionally activated polyethyleneglycol is a difunctionally activated polyethylene glycol.
 3. The processof claim 1, wherein the pellet is dispensed into a syringe prior toirradiation.
 4. The process of claim 1, wherein the radiation dosage iswithin the range of about 0.5 Mrad to about 4 Mrad.
 5. The process ofclaim 4, wherein the e-beam irradiation is within the range of about 1.5Mrad to about 2.5 Mrad.